Characterization of a dual biotin tag for improved single stranded DNA production

Abstract

Generation of single-stranded DNA plays a key role in many biotechnology applications including production of aptamers, single strand conformation polymorphism, nuclease S1 mapping, pyrosequencing, genosensors, probe preparation and labelling, subtractive hybridization as well as nucleic acid sensing and microarrays. Several methods are available in the literature to produce single-stranded DNA from double-stranded DNA templates, such as extraction of the sense strand from denaturing gels, asymmetric PCR, use of streptavidin–biotin interaction, and some alternative methods, including enzymatic digestion of the negative strand by either lambda exonuclease or T7 Gene 6 exonuclease. In this report, a detailed characterization of a dual biotin tag method to generate single-stranded DNA from the random oligonucleotide library is presented. Unlike the traditional streptavidin–biotin method that uses single biotin tagged molecules during separation, this novel technique is based on a dual biotin molecule covalently attached to the 5′ end of the negative strand. The improved technique takes less than one hour as a consequence of the eliminated alkali treatment step, which makes this procedure the shortest procedure described in the literature so far for single-stranded DNA production. The method can achieve a single-stranded DNA yield around 75% from the corresponding DNA template in Tris–HCl buffer. A number of parameters, such as the effect of different elution buffers and heat treatments, spontaneous release of streptavidin from the magnetic bead surface, loss of beads during consecutive washes, aggregation of the beads, were investigated to reveal the optimal conditions for single-stranded DNA production. FTIR, DLS, SEM, and electrophoresis techniques were used for characterization studies.